Targeting the tlk1/nek1 axis in prostate cancer

ABSTRACT

A method of treating prostate cancer in a patient comprising administering to the patient a pharmaceutical composition including a first therapeutic including a TLK1B inhibitor, or a pharmaceutically acceptable salt, solvate, ester, amide, clathrate, stereoisomer, enantiomer, prodrug or analog thereof, and a second therapeutic including an antiandrogen or a pharmaceutically acceptable salt, solvate, ester, amide, clathrate, stereoisomer, enantiomer, prodrug or analog thereof.

CROSS REFERENCE TO RELATED APPLICATIONS/PRIORITY

The present invention claims priority to U.S. Provisional PatentApplication No. 62/825,800 filed Mar. 29, Day, 2019, which isincorporated by reference into the present disclosure as if fullyrestated herein. Any conflict between the incorporated material and thespecific teachings of this disclosure shall be resolved in favor of thelatter. Likewise, any conflict between an art-understood definition of aword or phrase and a definition of the word or phrase as specificallytaught in this disclosure shall be resolved in favor of the latter.

BACKGROUND

Prostate cancer (PCa) is diagnosed in over 200,000 men each year in theUS and accounts for one in four individuals after adjustment for age.Androgen deprivation therapy (ADT) is used to treat advanced PCa withsome initial benefits, but Castrate Resistant Prostate Cancer (CRPC)often ensues after 2-3 years. While there are some treatment modalitiesfor CRPC, resistance occurs after a few months and CRPC is currentlyincurable. Treatments that can significantly improve the benefits of ADTand delay progression have the highest potential of being rapidlyimplemented and result in a significantly better outcome for advancedPCa, but sufficient treatments have thus far eluded researchers andclinicians.

SUMMARY

Wherefore, it is an object of the present invention to overcome theabove-mentioned shortcomings and drawbacks associated with the currenttechnology.

The present invention relates to The present invention is related tocompositions and methods of treating prostate cancer in a patientcomprising administering to the patient a pharmaceutical compositionincluding a first therapeutic including a TLK1B inhibitor, or apharmaceutically acceptable salt, solvate, ester, amide, clathrate,stereoisomer, enantiomer, prodrug or analog thereof, and a secondtherapeutic including an antiandrogen or a pharmaceutically acceptablesalt, solvate, ester, amide, clathrate, stereoisomer, enantiomer,prodrug or analog thereof. According to a further embodiment the TLK1Binhibitor is a phenothiazine (PTH) antipsychotic. According to a furtherembodiment the PTH antipsychotic is one of Thioridazine (THD),Perphenazine (PPH), Trifloroperazine (TFP), and Promazine (PMZ),4-(2-(10H-phenothiazin-10-yl) ethyl) morpholine (J54), and10-methyl-10H-phenothiazine (J56). According to a further embodiment thePTH antipsychotic is J54. According to a further embodiment antiandrogenis one of bicalutamide, aminoglutethimide, ketoconazole, abirateroneacetate, enzalutamide, and apalutamide. According to a furtherembodiment the antiandrogen is bicalutamide. According to a furtherembodiment the TLK1B inhibitor is 4-(2-(10H-phenothiazin-10-yl) ethyl)morpholine (J54) and the antiandrogen is bicalutamide. According to afurther embodiment the patient is human. According to a furtherembodiment the patient is one of chemically and surgically castrated.

The present invention is further related to methods preparing and/orusing pharmaceutical composition and pharmaceutical compositionscomprising a first therapeutic including a TLK1B inhibitor, or apharmaceutically acceptable salt, solvate, ester, amide, clathrate,stereoisomer, enantiomer, prodrug or analog thereof, and a secondtherapeutic including an antiandrogen or a pharmaceutically acceptablesalt, solvate, ester, amide, clathrate, stereoisomer, enantiomer,prodrug or analog thereof. according to a further embodiment the TLK1Binhibitor is a phenothiazine (PTH) antipsychotic. According to a furtherembodiment the PTH antipsychotic is one of Thioridazine (THD),Perphenazine (PPH), Trifloroperazine (TFP), and Promazine (PMZ),4-(2-(10H-phenothiazin-10-yl) ethyl) morpholine (J54), and10-methyl-10H-phenothiazine (J56). According to a further embodiment thePTH antipsychotic is J54. According to a further embodiment antiandrogenis one of bicalutamide, aminoglutethimide, ketoconazole, abirateroneacetate, enzalutamide, and apalutamide. According to a furtherembodiment the antiandrogen is bicalutamide. According to a furtherembodiment the TLK1B inhibitor is 4-(2-(10H-phenothiazin-10-yl) ethyl)morpholine (J54) and the antiandrogen is bicalutamide. According to afurther embodiment, the pharmaceutical composition further comprises anexcipient. According to a further embodiment the pharmaceuticalcomposition is in the form of a tablet, a capsule, a liquid solution orsuspension, a powder, a liquid, or solid crystals.

The present invention further comprises a purified sample of a4-(2-(10H-phenothiazin-10-yl) ethyl) morpholine. According to a furtherembodiment the sample is a solid crystal.

The present invention further relates to targeting a specific liabilitythat incurs in Androgen Responsive PCa cells when shifted to ADT, byadding an inhibitor, such as J54, of the TLK1>Nek1>ATR>Chk1 DDR axis inorder to abrogate the checkpoint and promote apoptosis. J54 is a novel,structure-based inhibitor of TLK that holds much promise to preventprogression toward CRPC and death from PCa.

The present invention relates to pharmaceutical compositions of atherapeutic (e.g., a TLK1B inhibitor, an antiandrogen, and a combinationof each), or pharmaceutically acceptable salts, solvates, esters,amides, clathrates, stereoisomers, enantiomers, prodrugs or analogsthereof, and use of these compositions for the treatment of a PCa.

In some embodiments, the therapeutic, or a pharmaceutically acceptablesalt, solvate, or prodrug thereof, is administered as a pharmaceuticalcomposition that further includes a pharmaceutically acceptableexcipient.

In some embodiments, administration of the pharmaceutical composition toa human results in a peak plasma concentration of the therapeuticbetween 0.05 μM-10 μM (e.g., between 0.05 μM-5 μM).

In some embodiments, the peak plasma concentration of the therapeutic ismaintained for up to 14 hours. In other embodiments, the peak plasmaconcentration of the therapeutic is maintained for up to 1 hour.

In some embodiments, the condition is a PCa.

In certain embodiments, the PCa is mild to moderate PCa.

In further embodiments, the PCa is moderate to severe PCa.

In other embodiments, the therapeutic is administered at a dose that isbetween 0.05 mg-5 mg/kg weight of the human.

In certain embodiments, the pharmaceutical composition is formulated fororal administration.

In other embodiments, the pharmaceutical composition is formulated forextended release.

In still other embodiments, the pharmaceutical composition is formulatedfor immediate release.

In some embodiments, the pharmaceutical composition is administeredconcurrently with one or more additional therapeutic agents for thetreatment or prevention of the PCa.

In some embodiments, the therapeutic, or a pharmaceutically acceptablesalt, solvate, or prodrug thereof, is administered as a pharmaceuticalcomposition that further includes a pharmaceutically acceptableexcipient.

In some embodiments, administration of the pharmaceutical composition toa human results in a peak plasma concentration of the therapeuticbetween 0.05 μM-10 μM (e.g., between 0.05 μM-5 μM).

In some embodiments, the peak plasma concentration of the therapeutic ismaintained for up to 14 hours. In other embodiments, the peak plasmaconcentration of the therapeutic is maintained for up to 1 hour.

In other embodiments, the therapeutic is administered at a dose that isbetween 0.05 mg-5 mg/kg weight of the human.

In certain embodiments, the pharmaceutical composition is formulated fororal administration.

In other embodiments, the pharmaceutical composition is formulated forextended release.

In still other embodiments, the pharmaceutical composition is formulatedfor immediate release.

As used herein, the term “delayed release” includes a pharmaceuticalpreparation, e.g., an orally administered formulation, which passesthrough the stomach substantially intact and dissolves in the smalland/or large intestine (e.g., the colon). In some embodiments, delayedrelease of the active agent (e.g., a therapeutic as described herein)results from the use of an enteric coating of an oral medication (e.g.,an oral dosage form).

The term an “effective amount” of an agent, as used herein, is thatamount sufficient to effect beneficial or desired results, such asclinical results, and, as such, an “effective amount” depends upon thecontext in which it is being applied.

The terms “extended release” or “sustained release” interchangeablyinclude a drug formulation that provides for gradual release of a drugover an extended period of time, e.g., 6-12 hours or more, compared toan immediate release formulation of the same drug. Preferably, althoughnot necessarily, results in substantially constant blood levels of adrug over an extended time period that are within therapeutic levels andfall within a peak plasma concentration range that is between, forexample, 0.05-10 μM, 0.1-10 μM, 0.1-5.0 μM, or 0.1-1 μM.

As used herein, the terms “formulated for enteric release” and “entericformulation” include pharmaceutical compositions, e.g., oral dosageforms, for oral administration able to provide protection fromdissolution in the high acid (low pH) environment of the stomach.Enteric formulations can be obtained by, for example, incorporating intothe pharmaceutical composition a polymer resistant to dissolution ingastric juices. In some embodiments, the polymers have an optimum pH fordissolution in the range of approx. 5.0 to 7.0 (“pH sensitivepolymers”). Exemplary polymers include methacrylate acid copolymers thatare known by the trade name Eudragit® (e.g., Eudragit® L100, Eudragit®S100, Eudragit® L-30D, Eudragit® FS 30D, and Eudragit® L100-55),cellulose acetate phthalate, cellulose acetate trimellitiate, polyvinylacetate phthalate (e.g., Coateric®), hydroxyethylcellulose phthalate,hydroxypropyl methylcellulose phthalate, or shellac, or an aqueousdispersion thereof. Aqueous dispersions of these polymers includedispersions of cellulose acetate phthalate (Aquateric®) or shellac(e.g., MarCoat 125 and 125N). An enteric formulation reduces thepercentage of the administered dose released into the stomach by atleast 50%, 60%, 70%, 80%, 90%, ₉₅%, or even 98% in comparison to animmediate release formulation. Where such a polymer coats a tablet orcapsule, this coat is also referred to as an “enteric coating.”

The term “immediate release” includes where the agent (e.g.,therapeutic), as formulated in a unit dosage form, has a dissolutionrelease profile under in vitro conditions in which at least ₅₅%, 65%,75%, 85%, or 95% of the agent is released within the first two hours ofadministration to, e.g., a human. Desirably, the agent formulated in aunit dosage has a dissolution release profile under in vitro conditionsin which at least 50%, 65%, 75%, 85%, 90%, or 95% of the agent isreleased within the first 30 minutes, 45 minutes, or 60 minutes ofadministration.

The term “pharmaceutical composition,” as used herein, includes acomposition containing a compound described herein (e.g., a TLK1Binhibitor and antiandrogen combination, such as J54 and bicalutamide, orany pharmaceutically acceptable salt, solvate, or prodrug thereof),formulated with a pharmaceutically acceptable excipient, and typicallymanufactured or sold with the approval of a governmental regulatoryagency as part of a therapeutic regimen for the treatment of disease ina mammal.

Pharmaceutical compositions can be formulated, for example, for oraladministration in unit dosage form (e.g., a tablet, capsule, caplet,gelcap, or syrup); for topical administration (e.g., as a cream, gel,lotion, or ointment); for intravenous administration (e.g., as a sterilesolution free of particulate emboli and in a solvent system suitable forintravenous use); or in any other formulation described herein.

A “pharmaceutically acceptable excipient,” as used herein, includes anyingredient other than the compounds described herein (for example, avehicle capable of suspending or dissolving the active compound) andhaving the properties of being nontoxic and non-inflammatory in apatient. Excipients may include, for example: antiadherents,antioxidants, binders, coatings, compression aids, disintegrants, dyes(colors), emollients, emulsifiers, fillers (diluents), film formers orcoatings, flavors, fragrances, glidants (flow enhancers), lubricants,preservatives, printing inks, sorbents, suspensing or dispersing agents,sweeteners, or waters of hydration. Exemplary excipients include, butare not limited to: butylated hydroxytoluene (BHT), calcium carbonate,calcium phosphate (dibasic), calcium stearate, croscarmellose,cross-linked polyvinyl pyrrolidone, citric acid, crospovidone, cysteine,ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropylmethylcellulose, lactose, magnesium stearate, maltitol, maltose,mannitol, methionine, methylcellulose, methyl paraben, microcrystallinecellulose, polyethylene glycol, polyvinyl pyrrolidone, povidone,pregelatinized starch, propyl paraben, retinyl palmitate, shellac,silicon dioxide, sodium carboxymethyl cellulose, sodium citrate, sodiumstarch glycolate, sorbitol, starch (corn), stearic acid, stearic acid,sucrose, talc, titanium dioxide, vitamin A, vitamin E, vitamin C, andxylitol.

The term “pharmaceutically acceptable prodrugs” as used herein, includesthose prodrugs of the compounds of the present invention which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of humans and animals with undue toxicity, irritation,allergic response, and the like, commensurate with a reasonablebenefit/risk ratio, and effective for their intended use, as well as thezwitterionic forms, where possible, of the compounds of the invention.

The term “pharmaceutically acceptable salt,” as use herein, includesthose salts which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of humans and animalswithout undue toxicity, irritation, allergic response and the like andare commensurate with a reasonable benefit/risk ratio. Pharmaceuticallyacceptable salts are well known in the art. The salts can be prepared insitu during the final isolation and purification of the compounds of theinvention or separately by reacting the free base group with a suitableorganic or inorganic acid. Representative acid addition salts includeacetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate,benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate,citrate, cyclopentanepropionate, digluconate, dodecylsulfate,ethanesulfonate, fumarate, glucoheptonate, glycerophosphate,hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride,hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate,lauryl sulfate, malate, maleate, malonate, methanesulfonate,2-naphthalenesulfonate, nicotinate, oleate, oxalate, palmitate, pamoate,pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate,propionate, stearate, succinate, sulfate, tartrate, thiocyanate,toluenesulfonate, undecanoate, valerate salts, and the like.Representative alkali or alkaline earth metal salts include sodium,lithium, potassium, calcium, magnesium, and the like, as well asnontoxic ammonium, quaternary ammonium, and amine cations, including,but not limited to ammonium, tetramethylammonium, tetraethylammonium,methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine,and the like.

The terms “pharmaceutically acceptable solvate” or “solvate,” as usedherein, includes a compound of the invention wherein molecules of asuitable solvent are incorporated in the crystal lattice. A suitablesolvent is physiologically tolerable at the administered dose. Forexample, solvates may be prepared by crystallization, recrystallization,or precipitation from a solution that includes organic solvents, water,or a mixture thereof. Examples of suitable solvents are ethanol, water(for example, mono-, di-, and tri-hydrates), N-methylpyrrolidinone(NMP), dimethyl sulfoxide (DMSO), N,N′-dimethylformamide (DMF),N,N′-dimethylacetamide (DMAC), 1,3-dimethyl-2-imidazolidinone (DMEU),1,3-dimethyl-3,4,5,6-tetrahydro-2-(1H)-pyrimidinone (DMPU), acetonitrile(ACN), propylene glycol, ethyl acetate, benzyl alcohol, 2-pyrrolidone,benzyl benzoate, and the like. When water is the solvent, the solvate isreferred to as a “hydrate.”

The term “prevent,” as used herein, includes prophylactic treatment ortreatment that prevents one or more symptoms or conditions of a disease,disorder, or conditions described herein (e.g., PCa). Treatment can beinitiated, for example, prior to (“pre-exposure prophylaxis”) orfollowing (“post-exposure prophylaxis”) an event that precedes the onsetof the disease, disorder, or conditions. Treatment that includesadministration of a compound of the invention, or a pharmaceuticalcomposition thereof, can be acute, short-term, or chronic. The dosesadministered may be varied during the course of preventive treatment.

The term “prodrug,” as used herein, includes compounds which are rapidlytransformed in vivo to the parent compound of the above formula.Prodrugs also encompass bioequivalent compounds that, when administeredto a human, lead to the in vivo formation of therapeutic. Preferably,prodrugs of the compounds of the present invention are pharmaceuticallyacceptable.

As used herein, and as well understood in the art, “treatment” includesan approach for obtaining beneficial or desired results, such asclinical results. Beneficial or desired results can include, but are notlimited to, alleviation or amelioration of one or more symptoms orconditions; diminishment of extent of disease, disorder, or condition;stabilized (i.e. not worsening) state of disease, disorder, orcondition; preventing spread of disease, disorder, or condition; delayor slowing the progress of the disease, disorder, or condition;amelioration or palliation of the disease, disorder, or condition; andremission (whether partial or total), whether detectable orundetectable. “Treatment” can also mean prolonging survival as comparedto expected survival if not receiving treatment. As used herein, theterms “treating” and “treatment” can also include delaying the onset of,impeding or reversing the progress of, or alleviating either the diseaseor condition to which the term applies, or one or more symptoms of suchdisease or condition.

The term “unit dosage forms” includes physically discrete units suitableas unitary dosages for human subjects and other mammals, each unitcontaining a predetermined quantity of active material calculated toproduce the desired therapeutic effect, in association with any suitablepharmaceutical excipient or excipients.

As used herein, the term “plasma concentration” includes the amount oftherapeutic present in the plasma of a treated subject (e.g., asmeasured in a rabbit using an assay described below or in a human).

Various objects, features, aspects, and advantages of the presentinvention will become more apparent from the following detaileddescription of preferred embodiments of the invention, along with theaccompanying drawings in which like numerals represent like components.The present invention may address one or more of the problems anddeficiencies of the current technology discussed above. However, it iscontemplated that the invention may prove useful in addressing otherproblems and deficiencies in a number of technical areas. Therefore theclaimed invention should not necessarily be construed as limited toaddressing any of the particular problems or deficiencies discussedherein.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

The accompanying drawings, which are incorporated in and constitute apart of the specification, illustrate various embodiments of theinvention and together with the general description of the inventiongiven above and the detailed description of the drawings given below,serve to explain the principles of the invention. It is to beappreciated that the accompanying drawings are not necessarily to scalesince the emphasis is instead placed on illustrating the principles ofthe invention. The invention will now be described, by way of example,with reference to the accompanying drawings in which:

FIGS. 1A-1E show that bicalutamide in combination with THD suppressesgrowth of LNCaP xenografts via pNek1-pATR-pChk1 DDR Pathway. FIG. 1Ashows tumor volume for LNCaP Xenograft model treated withTHD/Bicalutamide or combination by I.P. injections. FIG. 1B shows tumorweight for LNCaP Xenograft model treated with THD/Bicalutamide orcombination. FIG. 1C shows Western blot on tissue lysate from xenografttumors for Phospho-protein of DDR pathways. FIG. 1D shows representativeLNCaP xenograft tumors pic. Below is graph for tumor weight ofrespective groups. FIG. 1E shows representative images ofImmunohistochemistry for p-NEK1 on control/THD/Bicalutamide orcombination (Scale40×). The adjacent graph is quantitation.

FIG. 1F-1I show detailed views of homology model or Human TLK1 kinasedomain. In FIG. 1F, the model shows important regulatory features lyingin the N and C-lobes. FIG. 1G shows the hinge region connecting N andG-lobes. FIG. 1H shows a detailed view of DFG loop, Activation loop, P+1loop and the aF-helix. FIG. 1I shows the ATP binding site of the kinasedomain.

FIGS. 2A-2D show a Homology Model and Molecular Docking studies of newphenothiazine derivative (J54) with the modeled Human TLK1B kinasedomain. FIG. 2A depicts the homology model of TLK1 with active sites;ATP binding site, Allosteric sites 1 and 2. FIG. 2B depicts dock posefor compound J3-54 in the ATP-binding site. FIG. 2C depicts Dock posefor compound J3-54 in the allosteric binding site 1. FIG. 2D depictsDock pose for compound J3-54 in the binding site 2.

FIGS. 3A-3M show recombinant TLK1B purified to homogeneity. FIG. 3A isactive in kinase assays, FIG. 3B and was used in high-throughputinhibitor screening with a specific Nek1 peptide, with J54 shown in FIG.3C. FIGS. 3D-3E show Inhibitory curves of J54 and J56 compared to THDcarried out at 0.2 mM ATP or at different competitive concentrations.FIGS. 3F-3K show cytotoxicity and colony formation potential ofdifferent PCa cells, and FIGS. 3L and 3M show cell-cycle analysis ofLNCaP and TRAMP-C2 cells.

FIGS. 4A-4G show J54 treatment affects the expression of different DNAdamage response pathway proteins in combination with Bicalutamide. Shownare Western blots for p-Nek1, TLK1B, p-H2AX, c-PARP, Cleaved Caspase 8,PCNA, p21, p-ATR, and p-Chk1, in the absence of and treated with J54,Bicalutamide, and both J54 and Bicalutamide in LNCaP Cells and TRAMP-C2cells.

FIGS. 5A and 5B show Inhibition of TLK1/1B autophosphorylation by

PTH. FIG. 5A is a graph showing selected phenothiazines that were testedwith recombinant TLK1B in autokinase assays employing γ³²P-ATP anddetermined by TCA-precipitate counts (% activity compared to no drug.ED50 P<0.01). FIG. 5B shows an example of PTH inhibitor (PPH) that wastested in cultured cells by IP/autokinase/autoradiography. 293T cellswere incubated for 1 hour with PPH. TLK1 was immunoprecipitated,followed by auto-phosphorylation with γ-ATP³². The blots were thenprobed with TLK1 antiserum.

FIG. 6 is a graph showing that AI colonies do not develop with THD(concentration dependent). LNCap cells were plated in 6-well plates at4000 cells with bicalutamide (10 nM) to score developing AI colonies, or400 cells in control medium to monitor for general clonogenic inhibitionby THD.

FIGS. 7A and 7B are Western blots. FIG. 7A shows expression of TLK1B inLNCaP cells transferred to CSS-medium. Cells were grown in mediumcontaining FCS or transferred to CSS, for either 16 h (top), or during a4 h time-course (bottom). Where indicated, 20 nM rapamycin was alsoadded. FIG. 7B shows expression of TLK1B in NeoTag cells cultured withand w/o androgen.

FIG. 8A-FIG. 25 show further experiments into the present invention,with FIGS. 8A-8G show kinase assays, inhibitor screening, IC₅₀evaluation and competitive assays. Recombinant TLK1B purified tohomogeneity (FIG. 8A) is active in kinase assays (FIG. 8B) andphosphorylates a specific Nek1 peptide (FIG. 8C). FIG. 8D shows ATPdependence. FIG. 8E shows various compounds that were tested for invitro inhibitory effects, with J54 shown. FIGS. 8F and 8G showinhibitory curves of J54 and J56 compared to THD that were carried outat 0.1 mM ATP or at different competitive concentrations. Allexperiments were conducted in triplicates. The DiscoverRx ADP Hunter™(Eurofins DiscoverRx, Fremont, Calif., US) Kit was used to measure thegeneration of ADP resulting from kinase phosphorylation of substrate.

FIGS. 9A-9D show model building and molecular dynamics studies, withFIG. 9A showing a model of TLK1B kinase domain; FIG. 9B showing proteinRMSD for TLK1B over 1 μs simulation; FIG. 9C showing Ligand RMSD of J54(Black) and THD (Red) in post docking simulation for 100 ns; and FIG. 9Dshowing docking score and computed MM/GBSA binding free energy.

FIGS. 10A and 10B show molecular docking and molecular dynamics studiesof J54 and THD with TLK1, with FIG. 10A showing interactions of J54 withthe active site of TLK1; FIG. 10B showing interactions of THD with theactive site of TLK1.

FIGS. 11A-11E shows that J54 in combination with an anti-androgen(Bicalutamide) induces apoptosis in AS PCa cells. All experiments wereconducted in triplicates. FIG. 11A shows clonogenic assays of AS PCacells (VCaP, LNCaP, and TRAMP-C2) after treatment with BIC, J54, orcombination. The cells were grown for 2-3 weeks and stained with crystalviolet. All experiments were conducted in triplicates. FIG. 11 B showscell proliferation assays of the indicated cell lines incubated withdifferent concentrations of J54. The cell lines used were human “normal”RWPE-1, LNCaP, (LNCaP-derivative) C4-2B, 22RV1, DU14, PC3, and mouseTRAMP-C2. FIGS. 11C and 11D show cell proliferation of LNCaP andTRAMP-C2 were determined during a 3 days incubation with differentconcentration of J54 (MTS assay). All experiments were conducted intriplicates. FIG. 11E shows cell cycle analysis by PI-FACS of LNCaP andTRAMP-C2 cells incubated for 24 h with BIC, J54, or combination (5 μMeach). Representative analysis of two independent experiments are shown.

FIGS. 12A-12G are Western Blots of cell cycle and apoptotic indicators,which show that J54 in combination with Bicalutamide suppresses thecheckpoint activation markers and induces apoptotic markers. LNCaP,VCaP, and TRAMP-C2 cells from the four treatment groups as indicatedwere analyzed by WB for several indicators of DNA damage/apoptosis andmediators of cell cycle arrest. Representative analysis of twoindependent experiments for each cell line are shown

FIGS. 13A-13D show that the combination of bicalutamide and J54suppresses growth of LNCaP xenografts. FIG. 12A shows the time course oftumor growth of LNCaP cells xenografts in 4 treatment groups. Treatmentstarted 19 days after implantation when the tumors measure ˜200 mm³. Twoindependent experiment with 5 mice per group were carried out. J54 andBicalutamide was dissolved in DMSO and diluted in corn oil 1:10 andadministered IP bi-weekly. Sectioning and processing of the tissues werecarried out in the FWCC Histology Service, using automated processes andequipment to provide uniform and standardized results. Indirect labelingwas with ABC Elite: RTU Vectastain Elite Reagent, Vector # PK-7100; DAB:ImmPact DAB, Vector # SK-4105. Light counterstaining was done withhematoxylin. FIG. 13B shows tumor weights that were determined for allgroups at end of the treatment course. FIG. 13C shows examples of tumorsize at the end of the experiment. FIG. 13D shows representativesections from tumors resected from mice in the 4 treatment groupsanalyzed by IHC for pNek1. Note the weak pNek1 stain in the tumor frommice treated with J54, in contrast to the increase seen with BIC.

FIG. 14 shows an overlay of J54 and THD ligands within the active siteof TLK1, from various viewpoints.

FIGS. 15A-15C show docked pose of ligands to active site of D2 dopaminereceptor after 100 ns of MD simulation: (a) J54, (b) THD and (c)risperidone.

FIGS. 16A-16C show an H³NMR and MS of J54.

FIGS. 17A-17C show an H³NMR and MS of J3-56.

FIGS. 18A-18C show pharmacokinetics plasma distribution of J54.

FIGS. 19A-19C show behavioral assays in C. elegans. In FIG. 15A, for thepharyngeal pumping assay, wild-type animals (N2 strain) were transferredto plates with diluted DMSO (Control) or drug (J-54 or trifluoperazine)at 160 μM final concentration for 90 min before quantifying pharyngealpumping see SI methods). The inventors counted visible movement of thegrinder (pharyngeal contractions) for 30 sec to obtain the pumping rate(N=45). Note that in pharyngeal pumping, J54 reduced the pumping ratebut to a lesser extent than a much lower concentration of TFP.Inhibition of pharyngeal pumping by trifluoperazine (TFP), a typical PTHantipsychotic, is attributed to its known activity as a dopamine andcalmodulin antagonist. In FIG. 15B. foraging, regulated by serotonin anddopamine, was studied in wild-type animals evaluated in the presence ofDMSO (Control) or J54 at a high dose of 160 μM vs. TFP at 40 μM. Thenumber of omega turns (head touches body) and reversals were thencounted over the next 3 min to quantify search behavior for each group(N=13 per group). FIG. 15C shows reduction of dopamine-inducedimmobility. Dopamine produces immobility in C. elegans after 2-3 hr ofexposure. Antipsychotic drugs, such as haloperidol, that potently blockD2 dopamine receptors largely counteract the effects of dopamine, i.e.,more animals continue to move on plates that include dopamine plus drug.Wild-type animals were incubated on 60 agar plates with bacteria anddiluted DMSO (Control) or J54 or haloperidol at 160 μM finalconcentrations. After 1 hr on these plates, they were transferred to 60mm agar plates with bacteria that also contained dopamine (finalconcentration 25 mM) plus diluted DMSO (Control) or else J-54 orhaloperidol at final concentrations of 160 μM. After 3 hr on thedopamine plates, the inventors examined movement and counted animals asmoving if they traversed half their body length in either the forward orbackward direction during a 5-sec observation period. The inventors thencalculated the percentage of animals moving and repeated this experimentthree times to confirm the effects of drug. J54 was not toxic to wormseven after prolonged exposure.

FIG. 20A shows H&E stain of representative LNCaP tumors (from 3 mice),and FIG. 20B shows qRT-PCR quantitation of TLK1B mRNA expression (from 3mice per group in triplicate reactions).

FIG. 21 shows IHC analysis of markers of proliferation (Ki67), apoptosis(Cleaved PARP and Caspase 3), and presence of γH2AX (an indicator ofDSBs—Quantitation of the stained sections is shown on the side.Representative section from 3 mice per group are shown.

FIG. 22 is a table listing the chemical structure of J3-50, J3-51,J3-55, J3-65 and J3-66 compounds.

FIG. 23 is a table listing the antibodies used for Western blots andIHC.

FIG. 24 is a table showing cell cycle analysis.

FIG. 25 is a table showing the inhibitory effect of test substances onradioligand binding to two recombinant human dopamine receptors (DR2).

DETAILED DESCRIPTION

The present invention will be understood by reference to the followingdetailed description, which should be read in conjunction with theappended drawings. It is to be appreciated that the following detaileddescription of various embodiments is by way of example only and is notmeant to limit, in any way, the scope of the present invention. In thesummary above, in the following detailed description, in the claimsbelow, and in the accompanying drawings, reference is made to particularfeatures (including method steps) of the present invention. It is to beunderstood that the disclosure of the invention in this specificationincludes all possible combinations of such particular features, not justthose explicitly described. For example, where a particular feature isdisclosed in the context of a particular aspect or embodiment of theinvention or a particular claim, that feature can also be used, to theextent possible, in combination with and/or in the context of otherparticular aspects and embodiments of the invention, and in theinvention generally. The term “comprises” and grammatical equivalentsthereof are used herein to mean that other components, ingredients,steps, etc. are optionally present. For example, an article “comprising”(or “which comprises”) components A, B, and C can consist of (i.e.,contain only) components A, B, and C, or can contain not only componentsA, B, and C but also one or more other components. Where reference ismade herein to a method comprising two or more defined steps, thedefined steps can be carried out in any order or simultaneously (exceptwhere the context excludes that possibility), and the method can includeone or more other steps which are carried out before any of the definedsteps, between two of the defined steps, or after all the defined steps(except where the context excludes that possibility).

The term “at least” followed by a number is used herein to denote thestart of a range beginning with that number (which may be a range havingan upper limit or no upper limit, depending on the variable beingdefined). For example “at least 1” means 1 or more than 1. The term “atmost” followed by a number is used herein to denote the end of a rangeending with that number (which may be a range having 1 or 0 as its lowerlimit, or a range having no lower limit, depending upon the variablebeing defined). For example, “at most 4” means 4 or less than 4, and “atmost 40% means 40% or less than 40%. When, in this specification, arange is given as “(a first number) to (a second number)” or “(a firstnumber)-(a second number),” this means a range whose lower limit is thefirst number and whose upper limit is the second number. For example, 25to 100 mm means a range whose lower limit is 25 mm, and whose upperlimit is 100 mm. The embodiments set forth the below represent thenecessary information to enable those skilled in the art to practice theinvention and illustrate the best mode of practicing the invention. Inaddition, the invention does not require that all the advantageousfeatures and all the advantages need to be incorporated into everyembodiment of the invention.

Turning now to FIGS. 1A-21, a brief description concerning the variouscomponents of the present invention will now be briefly discussed. Afirst aspect of the present invention is the discovery that targetingthe TLK1/NEK1 axis with specific TLK1 inhibitors will be an effectivetherapy for PCa in combination with standard care, ADT. A second aspectof the present invention is the discovery of key compounds

The standard therapy for advanced Prostate Cancer (PCa) consists ofanti-androgens which provide respite from the disease progression, yetultimately fail and result in the incurable phase of the disease: mCRPC.Targeting PCa cells before their progression to mCRPC wouldsignificantly improve the outcome. Untoward toxicity limits thecombination therapies targeting the DNA Damage Response (DDR), and hencethe goal of clinical trials is to target the DDR more specifically.Androgen deprivation therapy (ADT) in LNCaP cells results in theincreased expression of TLK1B, a critical kinase upstream of NEK1 andATR, thereby mediating a DDR that typically causes a temporary cellcycle arrest of androgen-responsive PCa cells. Following the DNA damage,the addition of a TLK1 specific inhibitor, thioridazine (THD), impairsATR and Chk1 activation, establishing the existence of anADT>TLK1>NEK1>ATR>Chk1 DDR pathway, while its abrogation, leads toapoptosis. However, THD is a known anti-psychotic and has undesirableside-effects. Hence, there is a compelling need to design and developnext-generation TLK1 inhibitors to circumvent the adverse effects andadvance them in the clinic.

Our experimental data revealed that the pATR, pChk1, pNEK1, Ki-67 andPCNA were remarkably inhibited when treated with THD in combination withan anti-androgen drug, Bicalutamide (BIC). Moreover, it also inducedapoptosis and increased DNA damage as demonstrated by the cleaved PARP,Caspase 3 and γH2AX levels respectively. The new inhibitor screeningassay showed J54 compound to be most potent and inhibitory with alogIC₅₀ of 1.1 μM. J54 binds to the protein's allosteric sitenoncompetitively with ATP and interacts with His504 and Gly630 with acorresponding docking score of −6.736. J54 is found to be non-toxic tonormal cells and also suppresses the growth of androgen-dependentcolonies of LNCaP cells cultured with BIC.

The inventors' work evidences that targeting the TLK1/NEK1 axis withspecific TLK1 inhibitors will be an effective therapy for PCa incombination with standard care, ADT.

We performed immunoblotting of the tumor tissue phosphoproteins (pATR,pChk1 and pNEK1) and immunohistochemistry analysis of the tissuesections from the LNCaP xenograft models. To identify and develop newpotent inhibitors against TLK1, the inventors employed an in-silicohomology modelling and molecular docking approach. Based on theprotein-ligand binding interactions and the docking score, a handful ofcompounds were shortlisted, synthesised and screened for the TLK1inhibition potential in-vitro and using cell-based assays.

The Tousled Like kinase 1 (TLK1) is involved in the DNA damage responseand repair, and mitotic segregation of chromosomes. The inventorsidentified TLK1B, a splice variant of TLK1, as an important effector ofchemo-resistance that is often overexpressed in PCa cell lines andbiopsies, and its expression was identified as a key driver of PCa. Theinventors have identified several specific inhibitors of TLK1B and havedemonstrated that they sensitize PCa cells to killing by doxorubicin intissue culture or as mouse xenografts. However, the mechanism of actionof TLK1B remained to be largely elucidated, since most of its substrateshave not been identified. The inventors have now identified the proteometarget of TLK1B, and have identified an important new interaction withNEK1, a member of the NIMA related kinases, which is involved in the DNADamage Response (DDR) and Chk1 activation. Hence, the inventorshypothesize that TLK1/NEK1 axis is a critical target for therapy, andparticularly to enhance response to ADT. The inventors hypothesized thatinhibition of the TLK1/NEK1 axis will suppress the DDR and cell cyclearrest elicited by ADT, and result in excess replication-induced DNAdamage, leading to death of PCa cells. The inventors have acquiredevidence for this by demonstrating that inhibition of TLK1B withthioridazine (a phenothiazine antipsychotic in a class of specific TLKinhibitors) resulted in: 1) Complete inhibition of the conversion ofLNCaP cells to androgen independent growth after three weeks of culturewith bicalutamide (antiandrogen). 2) Inhibition of NEK1 activation thatoccurs in LNCaP cells cultured in charcoal-stripped serum (CSS)concomitant with increased expression of TLK1B. The inventors also foundthat expression of the NEK1-T141A mutant that cannot be phosphorylatedby TLK1B resulted in loss of Chk1 phosphorylation/activation followingDNA damage (H2O2) and impaired checkpoint establishment, as theinventors had expected.

The inventors identified inhibitors of TLK1 and found several in theclass of phenothiazine (PTH) antipsychotics which the inventors proposeto repurpose for the treatment of PCa to improve response to ADT.Further delineation of the TLK1/NKE1 axis and its involvement insurvival of PCa cells following ADT can lead to the discovery of evenmore specific PTH inhibitors, which the inventors intend to pursue. Thetranslational potential of this work is immediate since it would not bedifficult to reposition a FDA-approved PTH in combination with ADT. Thisresearch combines novel basic findings with innovative use of a class ofknown drugs.

Identification of TLK inhibitors. The inventors have screened thePrestwick Library, partially the ChemDiv, and two proprietary libraries(10,000 compounds) for inhibitors of TLK with a high throughput screenwith recombinant TLK1B and a Rad9 peptide substrate. The inventorsidentified four strong inhibitors in the class of PTH antipsychotics:Thioridazine (THD), Perphenazine (PPH), Trifloroperazine (TFP), andPromazine (PMZ), that block the dopamine D2 receptor in the brain. Theinhibition of autokinase activity was confirmed by TCA-precipitablecounts with γ32P-ATP (FIG. 5A). The drugs worked specifically at low μMconcentration after immunoprecipitation (IP) of TLK1 from cells, andthey remained associated with the protein, retaining their inhibitioneven after removal (after IP the inventors did not add the drugs in thekinase reaction—FIG. 5B).

The inhibitors markedly increased sensitivity to doxorubicin that couldbe explained by inhibition of NHEJ, as shown by slower regression of DSBrepair foci (γH2AX) and delayed repair kinetics of a single DSBgenerated with HO endonuclease on a reporter system. The specificity ofTHD was tested on a kinome panel (456 humankinases—KinomeScan-DiscoverX), and no other kinase (except TLK2) in thepanel was inhibited, although THD may impact other cellular functions.The inventors also showed that the TLK inhibitors sensitize PCa cells tokilling with doxorubicin in vitro and in xenografts. The inventorstested three PTH (THD, PPH, TFP) at 15 μM on a panel of cell lines,including RWPE1 and normal immortalized hTERT-HME1 cells, and the PTHhad low toxicity in the absence of doxorubicin.

Inhibition of colony formation of LNCaP cells with combined ADT andThioridazine (THD). One of the inventors' key hypotheses is thattreatment of androgen responsive PCa cells with antiandrogens wouldresult in upregulation of the TLK1/NEK1 axis via activation of mTORpathway, and that this axis is critical for survival of the cancercells. The inventors have tested this by monitoring the inhibition ofemergence of Androgen Insensitive (AI) colonies following treatment ofLNCaP cells with bicalutamide (antiandrogen) and increasingconcentrations of THD (TLK inhibitor). AI colonies were scored afterthree weeks. At a concentration of 3-9 μM THD, which inhibits growthonly 50% in androgen containing medium (FCS), few or no AI coloniesemerged (FIG. 6).

Expression of the TLK1B splice variant is translationally induced by ADTin LNCaP cells. Prostate cancer is characterized by its dependence onthe androgen receptor (AR) and frequent activation of PI3K signaling.PI3K pathway inhibition activates AR signaling and AR inhibitionactivates mTOR and AKT signaling. Thus, these two oncogenic pathwaysregulate each other by reciprocal feedback. Inhibition of one activatesthe other, thereby maintaining tumor cell survival. In FIG. 6 theinventors show that passing LNCaP cells to charcoal-stripped serum (CSS)results in a rapid increase in the expression of the TLK1B splicevariant. This is suppressible by rapamycin, strongly suggesting that itis due to the activation of the AKT/mTOR/eIF4E pathway (FIG. 7A).

The response to ADT is rapid, suggesting that it is most likely at thelevel of TLK1B mRNA translation, which likely results in parallel inactivation of NEK1. The inventors propose that activation of theTLK1/NEK1 axis following ADT is a very important, early pro-survivalpathway to cope with the DNA damage that ensues. The mechanism isprobably more general since another androgen sensitive cell line(NeoTag) also upregulated the expression of TLK1B in the absence ofandrogen (FIG. 7B). Targeting the TLK1/NEK1 axis could result insuppression of CRPC emergence.

A medicinal product of one embodiment of the present invention is incomposition of matter whereby treatment with ADT (standard of care foradvanced prostate cancer) is combined with an inhibitor of the TLK1/NEK1axis, which is critical for survival of cancer cells before progressionto CRPC. The combination therapy may include repurposing ofphenothiazine antipsychotic inhibitors of TLK1 for the treatment ofandrogen-responsive PCa.

A competitive advantage of some embodiments of the present inventioninclude that DNA damage response (DDR) includes the activation ofnumerous cellular activities that prevent duplication of DNA lesions andmaintain genomic integrity, which is critical for the survival of normaland cancer cells. Specific genes involved in the DDR such as BRCA1/2 andP53 are mutated during prostate cancer progression increasing thegenomic instability of cancer cells. These events may render prostatecancer cells particularly sensitive to inhibition of specific DDRpathways, such as PARP in homologous recombination DNA repair and Chk1(target of Nek1) in cell cycle checkpoint and DNA repair, creatingopportunities for synthetic lethality or synergistic cytotoxicity.Recent reports highlight the critical role of androgen receptor (AR) asa regulator of DDR genes, providing a rationale for combining DNAdamaging agents or targeted DDR inhibitors with AR inhibition astreatment for aggressive disease. Despite this promise, PARP inhibitorshave not been effective for PCa and showed some effect only in tumorswith HR deficiency (BRCA1/2). Targeting the DNA DSB repair machinery(ATM and DNA-PK) has been successful in sensitizing PCa cells to IR anddoxorubicin, as the inventors have found by targeting TLK1. This istheoretical support for a drug combination (e.g., bicalutamide orabiraterone in combination with a TLK1B inhibitor) be successful andresult in significant competitive advantage over similar DDR-basedstrategies that have not worked very well.

A further development of the present invention, including the compoundsJ54 and J56, follow.

TLK1 Homology Model: Due to unavailability of crystal structure forTLK1, the inventors performed de novo homology modelling and preparedmodel structure for kinase domain of human TLK1 protein (FIG. 1F). Themodel shows structural features hallmark to the kinase domain, theR-spine is in correct position and forms a connecting bridge between Nand C-lobes. The R-spine is followed by the DFG loop (Asp607, Phe608 andGly609) in the C-lobe. The hinge region is formed by residuesLeu538-Asn544, which allows the slight open and close motion between thetwo domains. The plausible ATP-binding site lies in the N-lobe and isformed by the residues like Leu462, Gly463, Ala483, Va1484, Leu538,Leu594, and Va1595 as shown in FIG. 1G. The activation loop is extendedcontinuation of the T-loop located in the C-lobe, it is considered as animportant regulatory element in the protein kinase, the T-loop continuesto the P+1 loop and the αF-helix (FIG. 1H).

To investigate the stability of the model, the inventors performed aclassical molecular dynamics simulation in explicit solvent in 0.1M slatconcentration for 500 ns at 300 K. The root-mean-square displacement(RMSD) in heavy atom positions converged to 4.5 Å after 500 ns,maintaining the predicted overall fold of the protein. The final TLK1structure from the simulation produced good validation scores, with aMolProbilty score of 1.49 and QMEAN score of −1.26. It was found to behighly accurate in the Ramachandran Plot with majority of residues (98%)were found in the favored and allowed region.

Three distinct active sites were identified by the SiteFinder andfavorable pockets in MD-refined structure for performing moleculardocking studies (FIG. 2A). The first site was obviously the ATP bindingsite, remaining two are identified as the allosteric site 1 and 2. Theallosteric site 1 is adjacent to the ATP binding site in the N-lobe,whereas the second allosteric site is found buried in the C-lobe.

The favorable pockets in the MD-refined TLK1 structure is shown in FIG.2A, The allosteric pocket 2 contained 198 spheres with a propensity forligand binding (PLB) score of 2.75, The active site 1 contained 108spheres with a PLB score of 2.43 and the allosteric site 3 contained 72spheres with PLB score of 1.80, the inventors note that a score ofgreater than 1.00 is considered highly druggable. Molecular dockingstudies were performed on all the three active sites. It was found thatcompound J3-54 could bind in all the three sites. In site 1 it docks atthe ATP binding site and has dock score of −8.2966 (Chemgauss4 scoringfunction), it also shows ionic interaction with Asp607. In a secondcase, the compound docks at allosteric site-1 with a better dockingscore of −10.0381. The morpholine head forming hydrogen bond with Glu499and ligand nucleus exposed towards solvent. The ligand binds to thesecond allosteric site but with docking score of 2.4059, the morpholinehead form a hydrogen bond with the Asp545. This suggests that thefavored site for interaction between the ligand and receptor would bethe allosteric region of the TLK1 protein.

Material and Methods: TLK1 homology modelling. Homology modelling wasperformed de novo via the ROBETTA full chain protein structurepredictive server, due to the absence of a template for comparativemodelling. The model was constructed by the hierarchical Ginzu screeningmethod. The modelled TLK1 structure was analyzed by a series ofvalidation methods for its reliability and consistency. The model wasfirst analyzed for its stereochemical quality using the MOE softwarepackage. It provided with analysis of steric problems within the proteinand high-accuracy Ramachandran Plot (RC-plot). The φ/ψ angles were wellwithin the allowed region. The majority of residues (98%) were found inthe favored and allowed region. Similar results were obtained on secondset of validation of the model using the MolProbity server. TheMolProbity score was 1.49, It showed the clash score for all atoms to be0.51 and six outlier rotamer. The energetic properties of TLK1 homologymodel was performed on the QMEAN web server. Qualitative Model Energyanalysis (QMEAN) was carried out of TLK1 homology model which revealed aQMEAN Value of −1.26.

Docking study. The molecular docking studies were performed on the

Openeye and MOE package. The homology model after molecular dynamicssimulation for 500 ns was subjected to molecular docking with compoundsfrom the ‘J3’ series of synthesized molecules. The energy minimized andpost-MD processed homology model was analyzed in MOE for determinationof active sites. Based on the active sites, the model was subjected tomolecular docking in the three different sites; ATP-binding site,allosteric site-1 and allosteric site-2. Docked poses were clustered andanalyzed for score and fit.

Molecular dynamics simulation. System preparation: MD simulations wereperformed using the AMBER16 software package. The TLK1 homology modelwas immersed in truncated octahedron of TIP3P water giving a total of10349 water molecules. Na+ and Cl− counter ions were added to neutralizethe system and achieve an ionic strength of 0.1 M. The ff14SB forcefield was used to model the protein.

Unbiased MD simulation: Simulations were performed using the pmemd.cudamodule. Simulations were run at 300 K using the Langevin thermostat witha collision frequency of 2 ps-1; and 1 atm using a Monte Carlo barostatwith volume exchange attempts every 100 fs. A 2 fs integration step wasemployed. Covalent bonds involving hydrogen were constrained usingSHAKE. A cutoff of 8 Å was used for short range nonbonded interactionswhilst long range electrostatics were treated using the particle meshEwald method. Equilibration consisted of rounds of NVT and NPTequilibration for 10 ns in total. Production MD run was performed for500 ns.

Continued J54/J56 Investigation

Through in-vitro kinase assays and molecular docking studies, theinventors report the synthesis and biological evaluation of a newphenothiazine with potent TLK1 inhibitory activity as a treatment forProstate Cancer still responsive to androgen-deprivation therapy. MostPCa deaths result from progressive failure in standard ADT, leading tometastatic castration-resistant PCa. Treatments that can suppress theconversion to mCRPC have the best potential to be rapidly implemented inthe clinics. ADT results in increased expression of TLK1B, a key kinaseupstream of NEK1 and ATR and mediating the DDR that typically results ina temporary cell cycle arrest of androgen responsive PCa cells, whileits abrogation leads to apoptosis. The inventors now studied J54 as newpotent inhibitor of this axis and as mediator of apoptosis in-vitro andin LNCaP xenografts, which has potential for clinical investigation incombination with ADT. J54 has low affinity for the dopamine receptor(DR2) in modelling studies and direct competitive radio-assays, and weakdetrimental behavioral effects in mice and C. elegans. Thus, J54 doesnot appear to have DR2 antagonistic side effects that can includecardiac arrhythmia.

J54, a novel potent inhibitor of TLK1, was synthesized based on insilico docking studies and kinase assays.

J54 leads to apoptosis of PCa cells in combination with Androgen

Deprivation Therapy in several PCa cells in LNCaP xenografts.

J54 has low affinity for recombinant dopamine receptors and lowantidopaminergic activity in animals.

TLK1B is selectively increased upon ADT, thereby providing a specifictarget for PCa cells rather than generally targeting the DDR, and thuscan mediate cancer specificity.

The novelty consists of a new specific inhibitor of TLK1 for PCa therapywithout the antidopaminergic side effects of previously studiedphenothiazines.

J3-54 (also called “J54”). The compound J54 is properly named4-(2-(10H-phenothiazin-10-yl) ethyl) morpholine. Description of thesynthesis of J54 is in the Appendix. The chemical structure of J54follows:

J3-56 (also called “J56”). The compound J56 is properly named10-methyl-10H-phenothiazine. Description of the synthesis of J56 is inthe Appendix. Its chemical structure of J56 follows.

Prostate Cancer (PCa) is a leading cause of morbidity and mortality ofmen in the western world. The standard of care for advanced PCa afterfailure of localized treatments is androgen-deprivation therapy (ADT)and anti-androgens, which provides respite from disease progression, butultimately fails resulting in the incurable phase of the disease: mCRPC.Treatments that can suppress the conversion to mCRPC have the bestpotential to improve outcome and be rapidly implemented in the clinics;and this requires a clear understanding of the process of PCa cells'mechanisms of adaptation to ADT. This process has been well studied inthe LNCaP cell line model, in which the inventors have recentlyelucidated some missing details. Androgen deprivation of LNCaP cellsresults in loss of AR function with a compensatory pro-survivalactivation of mTOR and concomitant implementation of a cell divisionarrest by activation of the DNA Damage Response (DDR) mediated byATR-Chk1 or ATM-Chk2. The DDR is likely activated due to the role playedby the AR as replication licensing factor in combination with the mTORdependent increased expression of TLK1B and resulting activation of theNek1>ATR>Chk1 axis. Additional work from the inventors' lab suggestedthat this may be a conserved nexus in additional cell models; in theTRAMP mouse; and probably in many patients, since the specificactivating phosphorylation of Nek1 by TLK1 correlates with the Gleasonscore. The resulting cell cycle arrest is a survival mechanism for PCacells, which remain quiescent until they reprogram and adapt to AndrogenIndependent (AI) growth. An attractive strategy to prevent this processwould be to bypass the cell cycle arrest via inhibition of ATM or ATR,causing the cells to undertake replication with damaged DNA that wouldcause mitotic catastrophe, a strategy that was in fact implemented inLNCaP treated concomitantly with bicalutamide and ATM inhibition.However, a limitation of this approach is how to make the inhibition ofATM or ATR specific to PCa cells to limit general toxicity. Theinventors have recently demonstrated that addition of a relativelyspecific inhibitor of TLK, thioridazine (THD), which the inventorsrepurposed for the blockage of the axis, results in fact in apoptosis ofLNCaP and TRAMP-C2 cells concomitantly treated with bicalutamide. Inaddition, it suppresses the late re-growth of PCa in the TRAMP mousefollowing castration. However, THD is a known anti-psychotic and hasundesirable side effects. Here the inventors describe J54, a new TLK1inhibitor, as an adjuvant to ADT for PCa.

Materials and Methods

Molecular Modelling, Docking, Molecular Dynamics Simulations and Free

Energy: Details were explained in details in section A of supplementarymaterial and methods.

Chemical synthesis of J54 and several different Phenothiazines andProtein expression and purification: Details were explained in section Bof supplementary of material and methods. The full-length, 65 KDarecombinant human TLK1B (hTLK1B) for the in-vitro experiments wasexpressed and purified from bacteria as described earlier.

Cell lines: All PCa cell lines were obtained from American Type CultureCollection (ATCC). Cell lines can be identified by Research ResourceIdentifier (RRID) as available in the ExPASy Cellosaurus database forLNCaP (RRID:CVCL_0395), VCaP (RRID:CVCL_2235), RWPE-1 (RRID:CVCL_3791),LNCaP C4-2B (RRID:CVCL_4784), 22Rv1 (RRID:CVCL_1045), DU145(RRID:CVCL_0105), PC-3 (RRID:CVCL_0035), TRAMP-C2 (RRID:CVCL_3615)).LNCaP, RWPE-1, LNCaP C4-2B, 22Rv1, DU145 and PC3 cell lines recentlyauthenticated using STR profiling within the last three years and freefrom mycoplasma, and cultured as per ATCC instructions. VCaP andTRAMP-C2 cell line were purchased recently. All cell line-culturerelated experiments were performed with mycoplasma-free cells.”

Antibodies: Antibodies used for present study is listed as FIG. 23.

LNCaP Xenograft Model and Immunohistochemistry (IHC): All animals usedin this study were approved by LSUHSC IACUC and following ARRIVEguidelines. Male NOD/SCID mice (5 per group) were purchased from Jacksonlabs. LNCaP cells were injected as described earlier. Mice were treatedIP bi-weekly with J54 (5 mg/kg body weight—dose estimation derived frompharmacokinetics study) and/or Bicalutamide (100 mg/kg).

Statistics: Significance between treatment groups (mean values and SDs)was performed by a one-way ANOVA followed by Tukey's multiplecomparisons tests using GraphPad Prism6 software (GraphPad SoftwareUSA). p-value<0.05 (*p<0.05; **p<0.01; ***p<0.001) was considered toindicating a statistically significant difference.

Availability of supporting data and material. All data generated oranalyzed during this study and its supplementary information files isavailable upon request. Limited amounts of J54 will be available uponrequest.

Results: A new PTH potent inhibitor of TLK1B. A few PTH antipsychoticswere identified in a screen the inventors conducted from a compoundslibrary as good inhibitors of TLK1. To find additional inhibitors,recombinant TLK1B was purified (FIG. 8A). The enzymatic activity wasdetermined using the ADP-Hunter reagents and a Nek1 peptide containingthe T141 target site. Properties of substrate (Nek1 peptide) and ATPdependence were determined (FIG. 8B-D), and in subsequent reactions withinhibitors used at 0.1 mM, which were found to be optimal. Severalstructurally similar compounds were synthesized as described in theAppendix (and see NMR and MS data FIGS. 10 and 11) and tested at 20 μM(FIGS. 8E and 8F). J3-54 (hereafter “J54”) and J3-56 (hereafter “J56”),both being PTH derivatives, were more inhibitory than Staurosporin,which is the standard pan-kinase inhibitor. The inventors continued theinventors' characterization of J54. The other synthesized compounds didnot show much inhibition even though several are PTH (FIG. 22). Theinventors have carried out some SAR studies and molecular modeling toexplain why, and for simplicity, the inventors show below a comparisonbetween THD and J54.

In-silico modeling studies of TLK1B with docked J54 or THD. A model ofthe TLK1B kinase domain was constructed by homology modeling using theROBETTA de novo protein structure prediction server. (see Methods inAppendix). The model was further refined by a 1 μs molecular dynamics(MD). The backbone RMSD of the structure stabilized after 100 nsalthough was subject to periodic RMSD shifts until 600 ns (FIG. 2A).Compounds J54 and THD were docked into the ATP site of the finalMD-refined model (FIGS. 9B and 9C). In the docked pose of compound J54,good interactions with the hinge region residues of TLK1 were exhibited;the morpholino head forms hydrogen bonds with Asp122, with a distance of1.68 Å from the Asp122 carboxylate Oδ to the ligand NH group (FIGS. 10Aand 9D). A 100 ns MD simulation of this TLK1-J54 complex indicated thepose is stable, further evident from a favorable total binding freeenergy ΔG_(bind)=−39.7 kcal/mol, computed by the MM/GBSA method (FIG.9D). THD was docked in the same TLK1 pocket but did not form hydrogenbonds with the protein (FIG. 14). MD of the complex indicated a markedlyhigher RMSD in ligand atoms, with greater fluctuation, than for the J45complex (FIG. 9C); the free energy of binding is 9.9 kcal/mol weaker(FIG. 9D). The inventors note that this in part could arise from themethylthio group of THD which prevents it from entering the hinge regionto the same extent as J54 (FIG. 14).

J54 in combination with anti-androgen (Bicalutamide) induces apoptosisin AS PCa cells. The inventors experimentally demonstrated that THD hassome growth inhibitory effects for several PCa cells lines, eitherandrogen sensitive (AS) or insensitive (AI). The inventors thus testedthe same panel of cells in androgen containing medium (FBS) byproliferation assays. The effect was a weak dose-dependent inhibitionwith maximal efficacy around 18 μM (FIG. 11B). Note however, that RWPE1(normal prostate) cell line was insensitive to J54, suggesting that J54may be a more targeted anti-cancer agent, or it is possible that theTLK1>Nek1 axis remains critical even for CRPC cells. In contrast tothese mild effects, combination treatment of the AS cell lines—LNCaP,VCaP and TRAMP-C2—with bicalutamide (BIC) and J54 resulted in 4-5-foldsuppression in colony formation (FIG. 11A, p=0.001). Note that VCaPcells were 60% inhibited by J54 alone, consistent with their elevatedreplication stress-driven DDR and checkpoint activation (FIG. 12E),which when suppressed with J54 could lead to apoptosis. Clonogenicassays are unable to distinguish the effects of a DNA damaging agent,which can result in either an impaired or delayed resumption of growth(cell division) or loss of viability due to increased killing of theinitial population. The inventors thus measured in LNCaP and TRAMP-C2cells the early change in cell number (MTT assay) over 72 h with J54alone. The results indicated an actual loss in cell counts in relationto the dose (FIGS. 11C and 11D, p=0.001). These results reinforced theinventors' main thesis that inhibiting the TLK1>Nek1 axis is mostly aneffective regimen for AS cells in combination with anti-androgens.Indeed, cell cycle analysis of LNCaP and TRAMP-C2 cells treated withBIC, J54, or combination for 24 h showed a strong increase in thefraction of apoptotic cells only in the combination group (FIG. 11E).Note also that, unlike cells treated with BIC that display anaccumulation of cells at G1/S and reduction of the S and G2 populations,cells treated with both BIC and J54 do not display the arrest(particularly no loss of the G2 cells), i.e., bypass of the G1checkpoint and consequent apoptosis (see FIG. 24).

J54 with Bicalutamide suppresses the checkpoint markers and inducesapoptotic markers. LNCaP, VCaP, and TRAMP-C2 are the main examples ofestablished PCa cell lines that are initially AS, but when maintained inADT condition, recapitulate the conversion to AI growth observed inpatients, and start growing again. In the initial phase of ADT, theyarrest cell division, primarily at the G1/S transition, and becomequiescent, which is a protective effect from undergoing replicationforks collapse upon inhibition of the licensing function of the AR.Since inhibition of the TLK1B>Nek1>ATR>Chk1 axis with THD results inbypass of the DDR and apoptosis, the inventors have tested the sameprocess with J54. First, the inventors confirmed that J54 (with orwithout BIC) causes a reduction in the p-Nek1-T141, the site ofactivation that is phosphorylated by TLK1, in all 3 AS cell lines (FIGS.12A-12G). The inventors were also able to reproduce that the expressionof TLK1B was induced in LNCaP and TRAMP-C2 following the addition ofBIC. The inventors experimentally showed that this is due to a mRNAtranslational effect caused by the compensatory increase in mTORactivity following ADT and suppression of AR signaling—note there was nochange in the TLK1B mRNA with BIC (FIG. 20B). Treatment with BICresulted in activation of the DDR, shown as an increase in p-ATR andp-Chk1 in LNCaP and TRAMP-C2. In contrast, suppression of p-Nek1 and itsactivity with J54 resulted in inhibition of the BIC-induced DDRactivation, as manifested by a decrease in p-ATR and p-Chk1 (FIGS.12A-12G). J54 alone caused a modest increase in p-ATR but not in Chk1 inLNCaP and TRAMP-C2, as was previously observed by the inventors withTHD, possibly due to a mild genotoxic effect. The VCaP cells were alittle different because even without any BIC, p-ATR and p-Chk1 werealready elevated. This has been observed before and attributed to aconstitutive DDR activation due to the TMPRSS2-ERG fusion in thesecells. Nonetheless, the addition of J54 to VCaP cells resulted inreduction of p-Nek1 and p-Chk1, indicating that it can suppress the DDRcheckpoint, whether activated constitutively or after BIC treatment(FIG. 12E). As the inventors previously demonstrated for inhibition ofTLK1 with THD, bypass of the BIC-induced checkpoint results inreplication forks collapse, generation of DSBs (indicated by thepresence of γH2AX), and leads to apoptosis (indicated by increasedcleaved caspase 3 and PARP—FIGS. 12A-12G). Likewise, in BIC+J54 treatedcells there was an increase in P21 expression: an indicator of theemergency activation of the P53>p21 pathway, and an effect previouslyobserved for BIC+THD.

Bicalutamide with J54 suppresses growth of LNCaP xenografts viasuppression of the TLK1B>pNek1 DDR pathway and promotes apoptosis. Theinventors sought to establish if the addition of J54 could suppress theresurgence of tumor growth of LNCaP xenografts and their latterconversion to CRPC. Following formation of sizeable tumors (˜200 mm³),castration or anti-androgens arrest the progression of LNCaP xenograftsfor some time (2-3 weeks), while subsequently the tumors start growingagain at an accelerated rate which is refractory to ADT (AI). Therefore,the inventors injected LNCaP cells in matrigel in both flanks ofNOD-SCID mice, and then randomly assigned them to four treatment groups(n=5 per group×2 independent experiments), as shown in FIGS. 13A-13C.The control group showed progressive exponential growth, and so did theBIC group after a 12 days lag following the beginning of BICadministration. Interestingly, treatment with J54 alone showedsignificant suppression of tumor growth (p=0.01) and tumor weight(p=0.001); whereas the combination (BIC+J54) resulted in completesuppression of tumor growth and weight (p=0.001) and actual regressionof the tumors compared to the starting size FIGS. 13A-13C. An IHCanalysis of the available excised tumors showed that the phosphorylationof Nek1-T141 was increased in BIC-treated group (FIG. 13D, p=0.001),consistent with a corresponding increase in TLK1B expression; it wassuppressed by concomitant administration of J54 (p=0.001), which isexpected to result in bypass of the DDR checkpoint and increasedapoptosis and corresponding markers. In fact, the combination treatmentshowed a strong increase in staining for cleaved PARP and Caspase 3, andγH2AX (a marker of DNA damage, p=0.001—FIG. 21). Tumors in thecombination also showed a reduction in the number of proliferative cellsby Ki67 staining (p=0.001), although this was lower also for the groupssingly treated with BIC and J54 (FIG. 21—Note that these tumors wereisolated at day 30 when they were still sizeable). The inventors shouldalso point out that the inventors treated the mice with J54 onlybi-weekly due to the favorable pharmacokinetics, where maximal plasmaconcentration of 100 ng/ml (35 μM) were reached 2 h after IP injection,and was still present at −6ng/ml (6 μM) after 24 h (FIG. 18A-18C).

J54 has low DR2 binding activity and behavioral effects in animalmodels. THD has strong antitumor effects in combination with ADT forAS-PCa according to the inventors' models. However, after ˜30 years ofuse for schizophrenia, this drug was withdrawn for treatment due toincreased risk of cardiac arrhythmia and extrapyramidal toxicity. Therepurposing of PTH anti-psychotics for cancer therapy was proposed bythe inventors even though their cellular targets have not beenidentified, as they were generally assumed by the inventors to worklargely through inhibition of dopamine receptors. The inventors wonderedif J54 has also anti-dopaminergic activity. The inventors thusconsidered the potential interaction with the D2 dopamine receptor(DR2). Compounds J54, THD and DR2 antagonist risperidone were studied bymolecular docking, MD and free energy calculations, based on therisperidone/DR2 crystal structure (PDB code 6CM4). The three ligandswere docked in the DR2 pocket (docking was able to reproduce theobserved pose of risperidone). The three complexes were solvated andsimulated via MD for 100 ns. Compound J54 binds stably in the activesite of the DR2, forming a hydrogen bond with Ser159 distance of 2.24 Å(FIG. 15A). THD and risperidone also complex with the DR2 via hydrogenbonds (FIG. 15A and 15C). Interestingly, the binding free energycomputed via MM/GBSA ranks J54 as the lowest affinity ligand, with aΔG_(bind) value of −29.0 (FIG. 9D). This is followed by THD (−39.0kcal/mol) and then risperidone (−53.4 kcal/mol). These free energycalculations suggest that J54 binds only weakly and has a lower affinitythan risperidone and THD towards DR2. To verify experimentally that J54is a weak DR2 antagonist, the inventors commissioned a competitiveradioassay using 7-Hydroxy DPAT, R-(+)-[3H] as a tracer and tworecombinant human dopamine receptors (D1 and D3). Positive controlantagonists were included [R(+)-SCH-23390 and(±)-7-Hydroxy-2-(di-n-propylamino) tetralin ((±)-7-OH-DPAT),]. In FIG.25, the inventors show the results obtained at effective concentrationsof 100 nM for J54 compared to THD, which confirmed that J54 is a veryweak antagonist for dopamine receptors. Note that in these competitivestudies with recombinant receptors, the antagonist (e.g, THD) istypically active in the 10 to 100 nM range (IC50-20 nM for THD), whereasfor DR2 antagonism for amelioration of psychotic conditions, circulatingconcentrations in plasma need to be ˜100 μM.

We then did a general assessment of mice following injections of J54.The inventors noticed no toxicity (even after gross inspection of organsat necropsy), no decrease in body weight, and no behavioral changes (nolethargy or extrapyramidal twitches sometime observed with THD). To geta better assessment of the possible behavioral effects of J54, theinventors used C. elegans that has a simple but complete nervous systemand has been well characterized for its responses to anti-psychoticdrugs, including actions at serotonin or dopamine receptors. In threestudies, J54 had much weaker behavioral effects then TFP or risperidone,attributable to DR2 activity (FIGS. 19A-19C). Note that theconcentration of these drugs on agar plates need to be significantlyhigher than in tissue culture medium for mammalian cells because of theprotective and poorly permeable cuticle in C. elegans.

DISCUSSION. Some PTH were found to be potent inhibitors of TLK1, and byinference TLK2 since these proteins must homo- and hetero-dimerize foractivation. Since not all PTH are good inhibitors of TLK1, it wascritical to first sort out in silico which compounds best fit in modeledbinding site. The inventors have now shown that J54 has higher affinitythan THD by MD analysis, and hence is more potent and evidence to bemore specific for TLK1. The inventors demonstrated that a TLK1inhibitor, THD, could synergize with ADT in promoting apoptosis on ASPCa cell lines in culture and in xenografts, and in the TRAMP mousemodel. This rationale was derived from the inventors' discovery thatTLK1 is an upstream activator of the Nek1>Atr>Chk1 axis, in conjunctionwith an original result that showed that TLK1B is translationallyincreased following AR suppression and the established compensatory mTORactivation. Abrogation of the TLK1>Nek1>ATR axis was expected to resultin bypass of the DDR checkpoint and thus promote mitotic catastrophe.This was also consistent with the known role of AR signaling inregulation of DNA repair in PCa, and synergistic killing with inhibitorsof DNA repair.

While it seems clear that certain PTH anti-psychotics correlate with adecreased risk of PCa development, as a downside, the use of THD andsome other PTH antipsychotics presents some risks and side effects.Therefore, the inventors have developed a second-generation TLKinhibitor that has lower affinity for the DR2. The potent dopamineantagonist activity of some PTH antipsychotics has been blamed for someof the lethargic and extrapyramidal effects, as well as for the cardiacarrhythmia. In silico, J54 showed much weaker binding to the DR2 thanTHD or risperidone, and it appeared to have little adverse behavioraleffects in mice and worms.

In this work, several PCa cell lines were studied for growth inhibitionby J54, and particularly the AS cell lines, LNCaP, VCaP, and TRAMP-C2,were sensitive to apoptosis when combined with an anti-androgen (BIC).The LNCaP model was also tested in xenografts and demonstratedremarkable tumor regression. In conclusion, the inventors suggest theuse of J54 as adjuvant therapy for PCa in conjunction withanti-androgens, as a safer and more potent inhibitor of this DDR axis,which the inventors believe is commonly activated during the initialphase of PCa cells' adaptation to ADT.

Conclusions. The regulation of the DDR by TLK1 through the Nek1>ATR>Chk1axis, and even more importantly its upregulation after ADT is a verynovel finding in the field of PCa research and therapy.

While a very large amount of work in PCa therapy has been devoted to thesearch for better anti-androgens, that is not the case for work devotedto combining ADT with targeting the known role of the AR in controllingthe DDR. The inventors' novel approach, which while counter-current tothe established views of standard of care for advanced PCa, is toabrogate the ADT-induced DDR checkpoint and cell cycle arrest, therebyforcing apoptosis of PCa cells still responsive to ADT.

While it seems clear that certain PTH anti-psychotics correlate with adecreased risk of PCa development, the use of THD and some other PTHanti-psychotics presents some risks and side effects. In particular anincreased risk for cardiac arrhythmia as a result of theiranti-dopaminergic activity and inhibition of hERG channels could impederepositioning of some of these PTH for the treatment of PCa due topotential concerns by regulatory agencies. J54 was designed and testedto be a weaker inhibitor of DR2, and the inventors in fact noticed ithad no apparent toxicity in mice, with no extrapyramidal twitches oraltered breathing after administration, and relatively weakanti-dopaminergic effects in C. elegans. Therefore, it is a bona fide,specific inhibitor of TLK1, and not the DR2, with superior efficacy andbetter side effects profile.

Pharmaceutical Compositions

The methods described herein can also include the administrations ofpharmaceutically acceptable compositions that include the therapeutic,or a pharmaceutically acceptable salt, solvate, or prodrug thereof. Whenemployed as pharmaceuticals, any of the present compounds can beadministered in the form of pharmaceutical compositions. Thesecompositions can be prepared in a manner well known in thepharmaceutical art, and can be administered by a variety of routes,depending upon whether local or systemic treatment is desired and uponthe area to be treated. Administration may be topical, parenteral,intravenous, intra-arterial, subcutaneous, intramuscular, intracranial,intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal,intracisternal, intraperitoneal, intranasal, aerosol, by suppositories,or oral administration.

This invention also includes pharmaceutical compositions which cancontain one or more pharmaceutically acceptable carriers. In making thepharmaceutical compositions of the invention, the active ingredient istypically mixed with an excipient, diluted by an excipient or enclosedwithin such a carrier in the form of, for example, a capsule, sachet,paper, or other container. When the excipient serves as a diluent, itcan be a solid, semisolid, or liquid material (e.g., normal saline),which acts as a vehicle, carrier or medium for the active ingredient.Thus, the compositions can be in the form of tablets, powders, lozenges,sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups,and soft and hard gelatin capsules. As is known in the art, the type ofdiluent can vary depending upon the intended route of administration.The resulting compositions can include additional agents, such aspreservatives.

The therapeutic agents of the invention can be administered alone, or ina mixture, in the presence of a pharmaceutically acceptable excipient orcarrier. The excipient or carrier is selected on the basis of the modeand route of administration. Suitable pharmaceutical carriers, as wellas pharmaceutical necessities for use in pharmaceutical formulations,are known. In preparing a formulation, the active compound can be milledto provide the appropriate particle size prior to combining with theother ingredients. If the active compound is substantially insoluble, itcan be milled to a particle size of less than 200 mesh. If the activecompound is substantially water soluble, the particle size can beadjusted by milling to provide a substantially uniform distribution inthe formulation, e.g. about 40 mesh.

Examples of suitable excipients are lactose, dextrose, sucrose,sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates,tragacanth, gelatin, calcium silicate, microcrystalline cellulose,polyvinylpyrrolidone, cellulose, water, syrup, and methyl cellulose. Theformulations can additionally include: lubricating agents such as talc,magnesium stearate, and mineral oil; wetting agents; emulsifying andsuspending agents; preserving agents such as methyl- andpropylhydroxy-benzoates; sweetening agents; and flavoring agents.

The methods described herein can include the administration of atherapeutic, or prodrugs or pharmaceutical compositions thereof, orother therapeutic agents. Exemplary therapeutics include those thatinhibit TLK1B (including of Thioridazine (THD), Perphenazine (PPH),Trifloroperazine (TFP), and Promazine (PMZ), J54, and J56) incombination with those that are antiandrogenic (including bicalutamide,aminoglutethimide, ketoconazole, abiraterone acetate, enzalutamide, andapalutamide).

The pharmaceutical compositions can be formulated so as to provideimmediate, extended, or delayed release of the active ingredient afteradministration to the patient by employing procedures known in the art.

The compositions can be formulated in a unit dosage form, each dosagecontaining, e.g., 0.1-500 mg of the active ingredients (TLK1B inhibitor,antiandrogenic, or both). For example, the dosages can contain fromabout 0.1 mg to about 50 mg, from about 0.1 mg to about 40 mg, fromabout 0.1 mg to about 20 mg, from about 0.1 mg to about 10 mg, fromabout 0.2 mg to about 20 mg, from about 0.3 mg to about 15 mg, fromabout 0.4 mg to about 10 mg, from about 0.5 mg to about 1 mg; from about0.5 mg to about 100 mg, from about 0.5 mg to about 50 mg, from about 0.5mg to about 30 mg, from about 0.5 mg to about 20 mg, from about 0.5 mgto about 10 mg, from about 0.5 mg to about 5 mg; from about 1 mg from toabout 50 mg, from about 1 mg to about 30 mg, from about 1 mg to about 20mg, from about 1 mg to about 10 mg, from about 1 mg to about 5 mg; fromabout 5 mg to about 50 mg, from about 5 mg to about 20 mg, from about 5mg to about 10 mg; from about 10 mg to about 100 mg, from about 20 mg toabout 200 mg, from about 30 mg to about 150 mg, from about 40 mg toabout 100 mg, from about 50 mg to about 100 mg of the active ingredient,from about 50 mg to about 300 mg, from about 50 mg to about 250 mg, fromabout 100 mg to about 300 mg, or , from about 100 mg to about 250 mg ofthe active ingredient. For preparing solid compositions such as tablets,the principal active ingredient is mixed with one or more pharmaceuticalexcipients to form a solid bulk formulation composition containing ahomogeneous mixture of a compound of the present invention. Whenreferring to these bulk formulation compositions as homogeneous, theactive ingredient is typically dispersed evenly throughout thecomposition so that the composition can be readily subdivided intoequally effective unit dosage forms such as tablets and capsules. Thissolid bulk formulation is then subdivided into unit dosage forms of thetype described above containing from, for example, 0.1 to about 500 mgof the active ingredient of the present invention.

Compositions for Oral Administration

The pharmaceutical compositions contemplated by the invention includethose formulated for oral administration (“oral dosage forms”). Oraldosage forms can be, for example, in the form of tablets, capsules, aliquid solution or suspension, a powder, or liquid or solid crystals,which contain the active ingredient(s) in a mixture with non-toxicpharmaceutically acceptable excipients. These excipients may be, forexample, inert diluents or fillers (e.g., sucrose, sorbitol, sugar,mannitol, microcrystalline cellulose, starches including potato starch,calcium carbonate, sodium chloride, lactose, calcium phosphate, calciumsulfate, or sodium phosphate); granulating and disintegrating agents(e.g., cellulose derivatives including microcrystalline cellulose,starches including potato starch, croscarmellose sodium, alginates, oralginic acid); binding agents (e.g., sucrose, glucose, sorbitol, acacia,alginic acid, sodium alginate, gelatin, starch, pregelatinized starch,microcrystalline cellulose, magnesium aluminum silicate,carboxymethylcellulose sodium, methylcellulose, hydroxypropylmethylcellulose, ethylcellulose, polyvinylpyrrolidone, or polyethyleneglycol); and lubricating agents, glidants, and antiadhesives (e.g.,magnesium stearate, zinc stearate, stearic acid, silicas, hydrogenatedvegetable oils, or talc). Other pharmaceutically acceptable excipientscan be colorants, flavoring agents, plasticizers, humectants, bufferingagents, and the like.

Formulations for oral administration may also be presented as chewabletablets, as hard gelatin capsules wherein the active ingredient is mixedwith an inert solid diluent (e.g., potato starch, lactose,microcrystalline cellulose, calcium carbonate, calcium phosphate orkaolin), or as soft gelatin capsules wherein the active ingredient ismixed with water or an oil medium, for example, peanut oil, liquidparaffin, or olive oil. Powders, granulates, and pellets may be preparedusing the ingredients mentioned above under tablets and capsules in aconventional manner using, e.g., a mixer, a fluid bed apparatus or aspray drying equipment.

Controlled release compositions for oral use may be constructed torelease the active drug by controlling the dissolution and/or thediffusion of the active drug substance. Any of a number of strategiescan be pursued in order to obtain controlled release and the targetedplasma concentration vs time profile. In one example, controlled releaseis obtained by appropriate selection of various formulation parametersand ingredients, including, e.g., various types of controlled releasecompositions and coatings. Thus, the drug is formulated with appropriateexcipients into a pharmaceutical composition that, upon administration,releases the drug in a controlled manner. Examples include single ormultiple unit tablet or capsule compositions, oil solutions,suspensions, emulsions, microcapsules, microspheres, nanoparticles,patches, and liposomes. In certain embodiments, compositions includebiodegradable, pH, and/or temperature-sensitive polymer coatings.

Dissolution or diffusion controlled release can be achieved byappropriate coating of a tablet, capsule, pellet, or granulateformulation of compounds, or by incorporating the compound into anappropriate matrix. A controlled release coating may include one or moreof the coating substances mentioned above and/or, e.g., shellac,beeswax, glycowax, castor wax, carnauba wax, stearyl alcohol, glycerylmonostearate, glyceryl distearate, glycerol palmitostearate,ethylcellulose, acrylic resins, dl-polylactic acid, cellulose acetatebutyrate, polyvinyl chloride, polyvinyl acetate, vinyl pyrrolidone,polyethylene, polymethacrylate, methylmethacrylate,2-hydroxymethacrylate, methacrylate hydrogels, 1,3 butylene glycol,ethylene glycol methacrylate, and/or polyethylene glycols. In acontrolled release matrix formulation, the matrix material may alsoinclude, e.g., hydrated methylcellulose, carnauba wax and stearylalcohol, carbopol 934, silicone, glyceryl tristearate, methylacrylate-methyl methacrylate, polyvinyl chloride, polyethylene, and/orhalogenated fluorocarbon.

The liquid forms in which the compounds and compositions of the presentinvention can be incorporated for administration orally include aqueoussolutions, suitably flavored syrups, aqueous or oil suspensions, andflavored emulsions with edible oils such as cottonseed oil, sesame oil,coconut oil, or peanut oil, as well as elixirs and similarpharmaceutical vehicles.

Compositions suitable for oral mucosal administration (e.g., buccal orsublingual administration) include tablets, lozenges, and pastilles,where the active ingredient is formulated with a carrier, such as sugar,acacia, tragacanth, or gelatin and glycerine.

Coatings: The pharmaceutical compositions formulated for oral delivery,such as tablets or capsules of the present invention can be coated orotherwise compounded to provide a dosage form affording the advantage ofdelayed or extended release. The coating may be adapted to release theactive drug substance in a predetermined pattern (e.g., in order toachieve a controlled release formulation) or it may be adapted not torelease the active drug substance until after passage of the stomach,e.g., by use of an enteric coating (e.g., polymers that are pH-sensitive(“pH controlled release”), polymers with a slow or pH-dependent rate ofswelling, dissolution or erosion (“time-controlled release”), polymersthat are degraded by enzymes (“enzyme-controlled release” or“biodegradable release”) and polymers that form firm layers that aredestroyed by an increase in pressure (“pressure-controlled release”)).Exemplary enteric coatings that can be used in the pharmaceuticalcompositions described herein include sugar coatings, film coatings(e.g., based on hydroxypropyl methylcellulose, methylcellulose, methylhydroxyethylcellulose, hydroxypropylcellulose, carboxymethylcellulose,acrylate copolymers, polyethylene glycols and/or polyvinylpyrrolidone),or coatings based on methacrylic acid copolymer, cellulose acetatephthalate, hydroxypropyl methylcellulose phthalate, hydroxypropylmethylcellulose acetate succinate, polyvinyl acetate phthalate, shellac,and/or ethylcellulose. Furthermore, a time delay material such as, forexample, glyceryl monostearate or glyceryl distearate, may be employed.

For example, the tablet or capsule can comprise an inner dosage and anouter dosage component, the latter being in the form of an envelope overthe former. The two components can be separated by an enteric layerwhich serves to resist disintegration in the stomach and permit theinner component to pass intact into the duodenum or to be delayed inrelease.

When an enteric coating is used, desirably, a substantial amount of thedrug is released in the lower gastrointestinal tract.

In addition to coatings that effect delayed or extended release, thesolid tablet compositions may include a coating adapted to protect thecomposition from unwanted chemical changes (e.g., chemical degradationprior to the release of the active drug substance). The coating may beapplied on the solid dosage form.

Parenteral Administration. Within the scope of the present invention arealso parenteral depot systems from biodegradable polymers. These systemsare injected or implanted into the muscle or subcutaneous tissue andrelease the incorporated drug over extended periods of time, rangingfrom several days to several months. Both the characteristics of thepolymer and the structure of the device can control the release kineticswhich can be either continuous or pulsatile. Polymer-based parenteraldepot systems can be classified as implants or microparticles. Theformer are cylindrical devices injected into the subcutaneous tissuewhereas the latter are defined as spherical particles in the range of10-100 μm. Extrusion, compression or injection molding are used tomanufacture implants whereas for microparticles, the phase separationmethod, the spray-drying technique and the water-in-oil-in-wateremulsion techniques are frequently employed. The most commonly usedbiodegradable polymers to form microparticles are polyesters from lacticand/or glycolic acid, e.g. poly(glycolic acid) and poly(L-lactic acid)(PLG/PLA microspheres). Of particular interest are in situ forming depotsystems, such as thermoplastic pastes and gelling systems formed bysolidification, by cooling, or due to the sol-gel transition,cross-linking systems and organogels formed by amphiphilic lipids.Examples of thermosensitive polymers used in the aforementioned systemsinclude, N-isopropylacrylamide, poloxamers (ethylene oxide and propyleneoxide block copolymers, such as poloxamer 188 and 407), poly(N-vinylcaprolactam), poly(siloethylene glycol), polyphosphazenes derivativesand PLGA-PEG-PLGA.

Mucosal Drug Delivery. Mucosal drug delivery (e.g., drug delivery viathe mucosal linings of the nasal, rectal, vaginal, ocular, or oralcavities) can also be used in the methods described herein. Methods fororal mucosal drug delivery include sublingual administration (viamucosal membranes lining the floor of the mouth), buccal administration(via mucosal membranes lining the cheeks), and local delivery.

Oral transmucosal absorption is generally rapid because of the richvascular supply to the mucosa and allows for a rapid rise in bloodconcentrations of the therapeutic.

For buccal administration, the compositions may take the form of, e.g.,tablets, lozenges, etc. formulated in a conventional manner. Permeationenhancers can also be used in buccal drug delivery. Exemplary enhancersinclude 23-lauryl ether, aprotinin, azone, benzalkonium chloride,cetylpyridinium chloride, cetyltrimethylammonium bromide, cyclodextrin,dextran sulfate, lauric acid, lysophosphatidylcholine, methol,methoxysalicylate, methyloleate, oleic acid, phosphatidylcholine,polyoxyethylene, polysorbate 80, sodium EDTA, sodium glycholate, sodiumglycodeoxycholate, sodium lauryl sulfate, sodium salicylate, sodiumtaurocholate, sodium taurodeoxycholate, sulfoxides, and alkylglycosides. Bioadhesive polymers have extensively been employed inbuccal drug delivery systems and include cyanoacrylate, polyacrylicacid, hydroxypropyl methylcellulose, and poly methacrylate polymers, aswell as hyaluronic acid and chitosan.

Liquid drug formulations (e.g., suitable for use with nebulizers andliquid spray devices and electrohydrodynamic (EHD) aerosol devices) canalso be used. Other methods of formulating liquid drug solutions orsuspension suitable for use in aerosol devices are known to those ofskill in the art.

Formulations for sublingual administration can also be used, includingpowders and aerosol formulations. Exemplary formulations include rapidlydisintegrating tablets and liquid-filled soft gelatin capsules.

Dosing Regimes. The present methods for treating PCas are carried out byadministering a therapeutic for a time and in an amount sufficient toresult in decreased tumor weight, decreased tumor growth, or decreasedin other incidence of PCa progression or presence.

The amount and frequency of administration of the compositions can varydepending on, for example, what is being administered, the state of thepatient, and the manner of administration. In therapeutic applications,compositions can be administered to a patient suffering from PCa in anamount sufficient to relieve or least partially relieve the symptoms ofthe PCa and its complications. The dosage is likely to depend on suchvariables as the type and extent of progression of the PCa, the severityof the PCa, the age, weight and general condition of the particularpatient, the relative biological efficacy of the composition selected,formulation of the excipient, the route of administration, and thejudgment of the attending clinician. Effective doses can be extrapolatedfrom dose- response curves derived from in vitro or animal model testsystem. An effective dose is a dose that produces a desirable clinicaloutcome by, for example, improving a sign or symptom of the PCa orslowing its progression.

The amount of therapeutic per dose can vary. For example, a subject canreceive from about 0.1 μg/kg to about 10,000 μg/kg. Generally, thetherapeutic is administered in an amount such that the peak plasmaconcentration ranges from 150 nM-250 μM.

Exemplary dosage amounts can fall between 0.1-5000 μg/kg, 100-1500μg/kg, 100-350 μg/kg, 340-750 μg/kg, or 750-1000 μg/kg. Exemplarydosages can 0.25, 0.5, 0.75, 1°, or 2 mg/kg. In another embodiment, theadministered dosage can range from 0.05-5 mmol of therapeutic (e.g.,0.089-3.9 mmol) or 0.1-50 μmol of therapeutic (e.g., 0.1-25 μmol or0.4-20 μmol).

The plasma concentration of therapeutic can also be measured accordingto methods known in the art. Exemplary peak plasma concentrations oftherapeutic can range from 0.05-10 μM, 0.1-10 μM, 0.1-5.0 μM, or 0.1-1μM. Alternatively, the average plasma levels of therapeutic can rangefrom 400-1200 μM (e.g., between 500-1000 μM) or between 50-250 μM (e.g.,between 40-200 μM). In some embodiments where sustained release of thedrug is desirable, the peak plasma concentrations (e.g., of therapeutic)may be maintained for 6-14 hours, e.g., for 6-12 or 6-10 hours. In otherembodiments where immediate release of the drug is desirable, the peakplasma concentration (e.g., of therapeutic) may be maintained for, e.g.,30 minutes.

The frequency of treatment may also vary. The subject can be treated oneor more times per day with therapeutic (e.g., once, twice, three, fouror more times) or every so-many hours (e.g., about every 2, 4, 6, 8, 12,or 24 hours). Preferably, the pharmaceutical composition is administered1 or 2 times per 24 hours. The time course of treatment may be ofvarying duration, e.g., for two, three, four, five, six, seven, eight,nine, ten or more days. For example, the treatment can be twice a dayfor three days, twice a day for seven days, twice a day for ten days.Treatment cycles can be repeated at intervals, for example weekly,bimonthly or monthly, which are separated by periods in which notreatment is given. The treatment can be a single treatment or can lastas long as the life span of the subject (e.g., many years).

Kits. Any of the pharmaceutical compositions of the invention describedherein can be used together with a set of instructions, i.e., to form akit. The kit may include instructions for use of the pharmaceuticalcompositions as a therapy as described herein. For example, theinstructions may provide dosing and therapeutic regimes for use of thecompounds of the invention to reduce symptoms and/or underlying cause ofthe PCa.

The invention illustratively disclosed herein suitably may explicitly bepracticed in the absence of any element which is not specificallydisclosed herein. While various embodiments of the present inventionhave been described in detail, it is apparent that various modificationsand alterations of those embodiments will occur to and be readilyapparent those skilled in the art. However, it is to be expresslyunderstood that such modifications and alterations are within the scopeand spirit of the present invention, as set forth in the appendedclaims. Further, the invention(s) described herein is capable of otherembodiments and of being practiced or of being carried out in variousother related ways. In addition, it is to be understood that thephraseology and terminology used herein is for the purpose ofdescription and should not be regarded as limiting. The use of“including,” “comprising,” or “having” and variations thereof herein ismeant to encompass the items listed thereafter and equivalents thereofas well as additional items while only the terms “consisting of” and“consisting only of” are to be construed in the limitative sense.

Wherefore, we claim:
 1. A method of treating prostate cancer in apatient comprising administering to the patient a pharmaceuticalcomposition including a first therapeutic including a TLK1B inhibitor,or a pharmaceutically acceptable salt, solvate, ester, amide, clathrate,stereoisomer, enantiomer, prodrug or analog thereof, and a secondtherapeutic including an antiandrogen or a pharmaceutically acceptablesalt, solvate, ester, amide, clathrate, stereoisomer, enantiomer,prodrug or analog thereof.
 2. The method of claim 1 wherein the TLK1Binhibitor is a phenothiazine (PTH) antipsychotic.
 3. The method of claim2 wherein the PTH antipsychotic is one of Thioridazine (THD),Perphenazine (PPH), Trifloroperazine (TFP), and Promazine (PMZ),4-(2-(10H-phenothiazin-10-yl) ethyl) morpholine (J54), and10-methyl-10H-phenothiazine (J56).
 4. The method of claim 3 wherein thePTH antipsychotic is J54.
 5. The method of claim 1 wherein theantiandrogen is one of bicalutamide, aminoglutethimide, ketoconazole,abiraterone acetate, enzalutamide, and apalutamide.
 6. The method ofclaim 5 wherein the antiandrogen is bicalutamide.
 7. The method of claim1 wherein the TLK1B inhibitor is 4-(2-(10H-phenothiazin-10-yl) ethyl)morpholine (J54) and the antiandrogen is bicalutamide.
 8. The method ofclaim 1 wherein the patient is human.
 9. The method of claim 1 whereinthe patient is one of chemically and surgically castrated.
 10. Apharmaceutical composition comprising: a first therapeutic including aTLK1B inhibitor, or a pharmaceutically acceptable salt, solvate, ester,amide, clathrate, stereoisomer, enantiomer, prodrug or analog thereof,and a second therapeutic including an antiandrogen or a pharmaceuticallyacceptable salt, solvate, ester, amide, clathrate, stereoisomer,enantiomer, prodrug or analog thereof.
 11. The pharmaceuticalcomposition of claim 10 wherein the TLK1B inhibitor is a phenothiazine(PTH) antipsychotic.
 12. The pharmaceutical composition of claim 11wherein the PTH antipsychotic is one of Thioridazine (THD), Perphenazine(PPH), Trifloroperazine (TFP), and Promazine (PMZ),4-(2-(10H-phenothiazin-10-yl) ethyl) morpholine (J54), and10-methyl-10H-phenothiazine (J56).
 13. The pharmaceutical composition ofclaim 12 wherein the PTH antipsychotic is J54.
 14. The pharmaceuticalcomposition of claim 10 wherein the antiandrogen is one of bicalutamide,aminoglutethimide, ketoconazole, abiraterone acetate, enzalutamide, andapalutamide.
 15. The pharmaceutical composition of claim 14 wherein theantiandrogen is bicalutamide.
 16. The pharmaceutical composition ofclaim 10 wherein the TLK1B inhibitor is 4-(2-(10H-phenothiazin-10-yl)ethyl) morpholine (J54) and the antiandrogen is bicalutamide.
 17. Thepharmaceutical composition of claim 10 further comprising an excipient.18. The pharmaceutical composition of claim 10 wherein thepharmaceutical composition is in the form of a tablet, a capsule, aliquid solution or suspension, a powder, a liquid, or solid crystals.19. A purified sample of a 4-(2-(10H-phenothiazin-10-yl) ethyl)morpholine.
 20. The purified sample of a 4-(2-(10H-phenothiazin-10-yl)ethyl) morpholine of claim 19 wherein the sample is a solid crystal.